Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis
Abstract
:1. Introduction
2. Results
2.1. Extracytoplasmic Stress-Response Regulators, RpoE and CpxR, Differentially Affect the Growth Rate of S. Enteritidis
2.2. CpxR Impacts Motility and Auto-Aggregation of S. Enteritidis
2.3. CpxR and CpxR/RpoE Have Profound Effects on Biofilm Formation
2.4. Effect of cpxR Deletion on the Biofilm Transcriptome
2.5. Effect of cpxR Deletion on the Transcriptome of Planktonic Cells
3. Discussion
4. Experimental Procedures
4.1. Bacterial Strains, Plasmids and Growth Conditions
4.2. Construction of ΔcpxR, ΔrpoE and ΔcpxR/ΔrpoE Salmonella Enteritidis Mutant Strains
4.3. Growth Assay
4.4. Swarming-Motility Assay
4.5. Auto-Aggregation Assay
4.6. Microtiter-Plate Biofilm Formation Assay
4.7. Flow-Cell Biofilm Formation Assay
4.8. Confocal Laser Scanning Microscopy (CLSM) and Biofilm Quantification
4.9. Preparation of Planktonic and Biofilm Samples for RNA Extraction
4.10. RNA-Seq Analysis
4.11. Validation of RNA-Seq Data Using Real-Time Polymerase Chain Reaction (PCR)
4.12. Experimental Replications and Bioinformatics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence (5′-3′) |
---|---|
Primers for rpoE and cpxR deletion | |
rpoE - Forward | ATG AGC GAG CAG TTA ACG GAC CAG GTC CTG GTT GAA CGG TGT AGG CTG GAG CTG CTT CG |
rpoE - Reverse | TCA ACG CCT GAT AAG CGG TTG AAC TTT ATT ATC AAT AGC CAT ATG AAT ATC CTC CTT AG |
cpxR - Forward | ATT AGC GAC GCC TGA TGA CGT AAT TTC TGC CTC GGA GGT ACG TAA ACA TGT AGG CTG GAG CTG CTT CG |
cpxR - Reverse | CCA GCG TCA ACC AGA AGA TGG CGA AGA TGC GCG CGG TTA AAC TTC CTA CAT ATG AAT ATC CTC CTT AG |
Primers for confirmation of rpoE and cpxR gene deletions | |
rpoE mutant F | GAC CTG TCT ACA ACA TGA CAA ACA |
rpoE mutant R | CGG ATC AGG TGA TAA CTC TCC CAG |
cpxR mutant F | CGC TTG CTC CCA AAA TCT TTT CTG |
cpxR mutant R | GTT GCT CTA TCA TCA ATC CCT GGC |
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Shetty, D.; Abrahante, J.E.; Chekabab, S.M.; Wu, X.; Korber, D.R.; Vidovic, S. Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis. Int. J. Mol. Sci. 2019, 20, 5146. https://doi.org/10.3390/ijms20205146
Shetty D, Abrahante JE, Chekabab SM, Wu X, Korber DR, Vidovic S. Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis. International Journal of Molecular Sciences. 2019; 20(20):5146. https://doi.org/10.3390/ijms20205146
Chicago/Turabian StyleShetty, Deeksha, Juan E. Abrahante, Samuel M. Chekabab, Xuxiaochen Wu, Darren R. Korber, and Sinisa Vidovic. 2019. "Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis" International Journal of Molecular Sciences 20, no. 20: 5146. https://doi.org/10.3390/ijms20205146